Characterizing gene expression at the single cell level can assist in understanding biological phenomena from development to cancer. Recent work has begun to address characterization of gene expression at the single cell level and has demonstrated several high-throughput methods for sequencing individual cells (see E. Z. Macosko, et al., Cell 161, 1202 (2015) and A. M. Klein, et al., Cell 161, 1187 (2015)). However, existing high-throughput single-cell sequencing methods do not generally preserve information about the spatial arrangement of cells in their original biological context. Thus, these methods may provide an incomplete picture of gene expression in complex multi-cellular systems.
Single-molecule fluorescence in situ hybridization (smFISH) techniques can be used to reveal RNA expression profiles in individual cells. Recent work has demonstrated that single-molecule RNA FISH can be used to sequentially label and image hundreds of mRNAs in individual cells (see E. Lubeck, et al., Nature Methods 11, 360 (2014); K. H. Chen, et al., Science 348, 412 (2015); and J. R. Moffitt, et al., PNAS 113(39), 11046 (2016)). However, smFISH may only reveal mRNAs for which specific sequences were designed, while sequencing can be used to measure the levels of all RNA present in the cell, even those with unexpected mutations or truncations. Moreover, smFISH techniques may not be likely to scale to the very large numbers of cells and transcripts that can be assayed with next generation sequencing.
Microdissection methods, such as laser capture microdissection, can be used for sequencing spatially defined groups of cells. However, this approach can require physical separation of the cells of interest and is not very high throughput. A recent study utilized a custom made oligonucleotide array to achieve high coverage gene expression measurements in tissue sections with spatial resolution. However, this strategy utilizes manufacture of a special array for every single measurement, the spatial resolution is determined by the array features, and this strategy does not scale to three-dimensional spatial mapping of tissue (see P. L. Stahl, et al., Science 353(6294), 78 (2016)).